JPH07308190A - Method for manufacturing thrombin - Google Patents

Abstract

PURPOSE:To obtain a thrombin having coagulating action from an inexpensive material industrially and advantageously, by treating a prothrombin-containing aqueous solution with a Ca salt in the absence of thromboplastin and plasma. CONSTITUTION:The supernatant after a cryoprecipitate is removed from human plasma is brought into contact with an anion exchanger such as DEAE-dextran. An adsorbed prothrombin complex is eluted to give a prothrombin-containing aqueous solution. Then the prothrombin-containing aqueous solution is treated with 3% aqueous solution of calcium chloride at 0-15 deg.C in the absence of thromboplastin and plasma for three days, adjusted to pH 7, treated with tri-N- butyl phosphate, epsilon-aminocaproic acid, etc., then brought into contact with an cation exchanger such as sulfopropyl-dextran and an adsorbed substance is eluted to give a thrombin having coagulating action by using an inexpensive material on an industrial scale.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing thrombin.

[0002]

BACKGROUND OF THE INVENTION Thrombin is a serine protease having a molecular weight of about 34,000 and an isoelectric point of about 7.1, and is a proteolytic enzyme that acts at the final stage of the blood coagulation process. That is, it acts on fibrinogen to produce fibrin, which causes a blood coagulation action. Therefore, thrombin is clinically used as a local hemostatic agent in the surgical field and as a hemostatic agent for upper gastrointestinal bleeding in the medical field. Thrombin exists in the form of prothrombin which is a precursor in the living body, and is produced by limited decomposition by activated factor X and the like. BACKGROUND ART Conventionally, thrombin has been produced by using human blood plasma as a raw material to first extract and purify prothrombin, and then allowing the obtained purified prothrombin to act on thromboplastin and the like.
That is, the conversion to thrombin is performed on purified prothrombin.

As described above, the production of thrombin begins with (1) purification of prothrombin as described above.
(2) conversion of prothrombin to thrombin, (3)
At present, it is manufactured through various processes such as purification of thrombin. In addition, various improved methods have been reported based on this method. For example, a method in which citrate-treated plasma is brought into contact with an anion exchanger, adsorbed prothrombin is converted to thrombin on the exchanger, and then eluted and recovered (US Pat. No. 5,138,389), plasma is treated with low temperature ethanol, A method is known in which prothrombin purified by treatment with an anion exchanger is converted into thrombin and further purified by treatment with a cation exchanger (JP-A-3-128398). Among them, as a method for converting into thrombin, a method using a snake venom other than the above thromboplastin, a method using high-concentration citrate (Japanese Patent Laid-Open No. 4-365481) and the like are known.

[0004]

By the way, thromboplastin prepared mainly from human placenta is used, but it is difficult to procure raw materials and it cannot always be said that a sufficient amount can be obtained. The same applies when using snake venom. On the other hand, the natural conversion method using high-concentration citrate is still at the laboratory scale, and has not been established as an industrial manufacturing method applicable to mass production.

In view of such circumstances, the present inventors have been working on the development of a method for producing thrombin on an industrial scale,
The present invention has been completed. That is, an object of the present invention is to provide a method for easily obtaining raw materials for conversion of prothrombin to thrombin and capable of producing and purifying thrombin on an industrial scale.

[0006]

The object of the present invention is achieved by a method for producing thrombin, which comprises treating an aqueous solution containing prothrombin with a Ca salt at 0 to 15 ° C. in the absence of thromboplastin and plasma. it can. The method for producing thrombin of the present invention, more specifically,
(1) Preparation of prothrombin-containing aqueous solution,
(2) conversion of prothrombin to thrombin, (3)
It consists of purification of thrombin.

(1) Preparation of Prothrombin-Containing Aqueous Solution The prothrombin-containing aqueous solution of the present invention is not particularly limited as long as it is plasma-derived prothrombin or a prothrombin complex or a fraction containing them. The prothrombin-containing aqueous solution of the present invention is a supernatant obtained by removing cryoprecipitate from plasma (decryoplasma), corn I + II + III.
It is prepared using the starting materials such as the fraction, the II + III fraction, and the III fraction. A known method may be used as a method of preparing the prothrombin-containing aqueous solution of the present invention from these starting materials. For example, a method utilizing the adsorptivity to an inorganic salt, anion exchanger treatment, affinity chromatography treatment, etc. are exemplified.

In the present invention, it is preferable to use a prothrombin complex. The prothrombin complex used here contains prothrombin (blood coagulation factor II), which is one of the blood coagulation factors, and other factors related to blood coagulation. Examples of the components contained together with prothrombin include blood coagulation factor VII, factor IX, factor X and the like. Further, it does not have to be a high-purity refined product and may be a crude product. The advantage of using the crude product is that it is not necessary to completely purify prothrombin before conversion to thrombin, so that the purification process is simplified and the cost is reduced. The origin of the prothrombin complex is not particularly limited.
Examples thereof include bovine-derived ones and human-derived ones, but preferably human-derived ones.

As a method for preparing the prothrombin complex (containing aqueous solution), known means can be used. For example, JP-A-62-10019 and JP-A-3-1283.
The method described in Japanese Patent Publication No. 98 can be used. Specifically, a method of treating plasma with an anion exchanger to prepare a prothrombin complex, a method of preparing a prothrombin complex from decryoplasma obtained by removing cryoprecipitate from plasma, and the like can be mentioned. As an anion exchanger, DEA
E type (eg DEAE-agarose, DEAE-dextran, DEAE-cellulose etc.), QAE type (eg QAE-agarose, QAE-dextran etc.)
Etc. The prothrombin complex obtained by the above means can be subjected to conversion treatment into thrombin in the state of the complex.

(2) Conversion of prothrombin to thrombin The prothrombin-containing aqueous solution of (1) is heated at 0 to 15 ° C.
C in the absence of thromboplastin and plasma
a Treat with salt. Examples of the Ca salt include calcium chloride, calcium hydroxide, calcium acetate and the like. Its concentration is about 10 to 50 mM. The prothrombin amount at that time is, for example, about 1 to 100 units / ml.
The treatment temperature is preferably 0 to 10 ° C. Processing time is 1-5
It is about a day. The pH of the solution is, for example, about 6-9. Furthermore, if necessary, known thrombin conversion methods may be combined. Preferably, the Ca salt-treated solution is subjected to autocatalytic reaction in a high-concentration citrate solution and spontaneously converted into thrombin. The conversion condition is a temperature of 20 to 40 ° C.
It is about 10 days. Examples of the citrate salt include sodium citrate. The concentration is 10-40 (w /
v) It is about%. The pH of the solution is, for example, about 6-9.

(3) Purification of thrombin, etc. The thrombin obtained in (2) is purified by a known method.
As a purification method, cation exchanger treatment, ammonium sulfate fractionation treatment,
Affinity chromatography treatment (for example, treatment using immobilized arginyl methyl ester or immobilized heparin) is exemplified. Further, it can be further purified by a known means such as dialysis, ultrafiltration or gel filtration.
By the above treatment, various contaminating proteins (eg, fibrinogen, fibrin, α ₁ globulin, α ₂ globulin, β globulin, γ globulin, protease, etc.) are substantially removed. The specific activity of such purified thrombin is about 500 to 1500 units / A ₂₈₀ .

The activity of thrombin can be determined by a usual measuring method. The method is preferably a method in which human plasma is used as a fibrinogen source and the coagulation time is measured by a BBL fibrometer or the like.
Specifically, 0.1 ml of the sample diluted with physiological saline at various ratios should be kept at 37 ° C for 2 minutes, and then 0.2 ml of plasma kept at 37 ° C should be added to measure the coagulation time. Can be sought by. The thrombin is formulated by a method known per se. That is, treatments such as addition of excipients, heating, sterilization, sterilization filtration, aliquot dispensing, lyophilization, etc. are carried out as necessary, and, for example, dry heat treatment (JP-A-62-81327), liquid Heating (JP-A-63-
243032), a treatment with trialkyl phosphates (EP Publication 378208) can be carried out if desired.

[0013]

According to the production method of the present invention, raw materials for converting prothrombin to thrombin can be easily obtained, can be efficiently prepared by a simple operation, and cost and labor can be sufficiently reduced. . Further, after the Ca salt treatment, a treatment with a known high-concentration citrate (Japanese Patent Application Laid-Open No. 4-365481) is performed, thereby reducing the treatment time (Ca salt treatment) without lowering the conversion efficiency from prothrombin to thrombin. Alternatively (including citrate treatment) can be shortened. The treatment conditions using high-concentration citrate include citrate concentration of 10 to 40 (w
/ V)% at 20 to 40 ° C. for about 1 to 10 days. Further, by using a prothrombin complex as prothrombin, labor required for complete purification to prothrombin can be omitted. Also, Ca salt treatment is 0
The efficiency of conversion into thrombin can be further improved by carrying out at -10 ° C. Therefore, the method of the present invention is a useful method suitable for industrial scale.

[0014]

EXAMPLES Examples and experimental examples are given in order to explain the present invention in more detail, but the present invention is not limited thereto. Example 1 Preparation of prothrombin and thrombin DEAE-dextran obtained by equilibrating 2 liters of decryoplasm obtained by freeze-thawing human plasma with a buffer solution (pH 7) consisting of 0.075 M sodium chloride and 0.01 M sodium citrate. (Protein name DEAE-Sephadex, A-50, manufactured by Pharmacia) to bring the prothrombin complex into a buffer solution (pH: 0.15 M sodium chloride and 0.01 M sodium citrate).
After washing with 7), 1M sodium chloride and 0.01M
Elution was performed with a buffer solution (pH 7) consisting of sodium citrate. The eluate was dialyzed, added with 1/10 amount of 3% calcium chloride, and treated at 5 ° C. for 3 days.

The pH was adjusted to 7, and tri-N-butyl phosphate (TNBP), Tween 80 and epsilon aminocaproic acid (EACA) were respectively added to final concentrations of 0.3% (w / v) and 1% (w / w). v) and 4% (w /
v), and heated at 30 ° C. for 6 hours. Next, the pH of the reaction solution was adjusted to 6.7, and sulfopropyl-dextran (trade name SP-) was equilibrated with a buffer solution (pH 6.7) consisting of 0.04 M sodium chloride and 0.02 M sodium citrate. Sephadex C-5
0, made by Pharmacia) to adsorb only thrombin. The column was buffered with 0.04 M sodium chloride and 0.02 M sodium citrate (pH 6.7), 0.1 M phosphate buffer (pH 6.
5), then 0.045M sodium citrate (pH
7) After washing with the solution, the adsorbed thrombin was eluted and isolated with a 0.1 M sodium citrate solution (pH 7). The eluate was concentrated with Pellicon (molecular weight cutoff 10,000: manufactured by Millipore), sterilized by filtration, and purified thrombin (specific activity 1000) was obtained.
Unit / A ₂₈₀ ) was prepared.

Example 2 Instead of treating with calcium chloride at 5 ° C. for 3 days, after treating with calcium chloride at 5 ° C. for 1 day, trisodium citrate was adjusted to 35 (w / v)%. The same treatment as in Example 1 was carried out except that prothrombin was converted to thrombin by treating the mixture with pH 8 at 37 ° C. for 2 days. Experimental Example 1 The relationship between temperature conditions during calcium chloride treatment and thrombin conversion efficiency was investigated. In Example 1, the temperature condition is 5 to 37.
The thrombin conversion efficiency was measured under each condition by setting the temperature to ° C. At the time of conversion, thromboplastin 1/1
0 amount was further added, and the thrombin conversion efficiency when treated at 15 ° C. for 3 hours was set to 100%. The results are shown in Table 1.

[0017]

[Table 1]

Experimental Example 2 The effect on the conversion efficiency of thrombin was examined between the case where only the Ca salt treatment was performed and the case where the Ca salt treatment and the high-concentration citrate treatment were combined. The results are shown in Table 2. The standard of conversion efficiency is the same as that of Experimental Example 1.

[0019]

[Table 2]

[0020]

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Shoichi Miyake 3-5-44, Miyakojima Nakadori, Miyakojima-ku, Osaka City, Osaka Prefecture Midori Cross Miyakojima Plant (72) Inventor Yuuhiko Matsumoto Miyakojima, Osaka City, Osaka Prefecture 3-5-44, Nakatsudori, Miyakojima, Tokyo Midori Cross Miyakojima Factory

Claims (5)

[Claims] 1. A prothrombin-containing aqueous solution at 0 to 15 ° C.
And a method for producing thrombin, which comprises treating with Ca salt in the absence of thromboplastin and plasma. 2. The above Ca salt treatment liquid is further added to 20 to 4
The method for producing thrombin according to claim 1, which is treated with 10 to 40 (w / v)% citrate at 0 ° C. for 1 to 10 days. 3. The method for producing thrombin according to claim 1, wherein the prothrombin is in the form of a prothrombin complex. 4. The method for producing thrombin according to claim 1, wherein the treatment with Ca salt is performed at 0 to 10 ° C. 5. The method for producing thrombin according to claim 1, wherein the treatment with Ca salt is carried out for 1 to 5 days.